Normal adult human tissues and cultured bronchial epithelial cells and fibroblasts exhibit 06-alkylguanine-DNA alkyltransferase activity in vitro by catalyzing the repair of the promutagenic alkylation lesion 06-methylguanine from DNA. The amount repaired by extracts of liver, peripheral lung, and colon extracts was proportional to the amount of extract protein. Repair of 06-methylguanine led to stoichiometric regeneration of guanine in the DNA and stoichiometric formation of S-methylcysteine in protein. Alkyltransferase activity varies in the different human tissues tested in the decreasing order of liver greater than colon greater than esophagus greater than peripheral lung greater than brain. Extracts of lung tissues, cultured human bronchial epithelial cells and fibroblasts had similar alkyltransferase activities. Various human tissues exhibit 2- to 10-fold higher alkyltransferase activity than corresponding rat tissues. Whereas the interindividual variation of the activity was 4- or 5-fold in 10 or more human lung and colon specimens, the interindividual variation in the inbred rat was less than 20%. The present results show that different human tissues and cells have a several-fold higher capacity to repair 06-methylguanine in DNA than rat tissues and that the repair process occurs via a mechanism similar to that previously shown in other mammalian cells and E. coli. Formaldehyde inhibits repair of 06-methylguanine and potentiates the mutagenicity of an alkylating agent, N-methyl-N-nitrosourea, in normal human fibroblasts. Because formaldehyde alone also causes mutations in human cells, formaldehyde may cause genotoxicity by a dual mechanism of directly damaging DNA and also inhibiting repair of mutagenic and carcinogenic DNA lesions caused by other chemical and physical carcinogens.